Delta opioid peptide (D-Ala2, D-Leu5)-enkephalin (DADLE) mitigates myocardial ischemia-reperfusion injury by inhibiting the TRAF6/NF-κB/NLRP3 pathway.

Objectives: This study aimed to assess the dose-dependent effect of DADLE and to explore its relationship with the TRAF6/NF-κB/NLRP3 pathway. Materials and Methods: After 45 min of ischemia, reperfusion was sustained for 24 hr in mice to establish the myocardial infarction model. DADLE was administered at doses of 0.25, 0.5, or 1 mg/kg to this model. TTC-Evans Blue double staining, HE staining, and Masson staining were conducted to evaluate myocardial injury. TUNEL staining was used to detect apoptosis. Western blotting and immunofluorescence staining were applied to measure levels of TRAF6, NF-κB p65, NLRP3, caspase-1, pro-caspase-1, and ASC. ELISA assays were used to assess TNF-α and IL-1β levels Results: DADLE at all three doses lessened the infarcted area compared with the PBS control. DADLE at 0.5 mg/kg was more efficacious than 0.25 and 1 mg/kg in reducing the infarcted size, pathological scores, and fibrosis. DADLE effectively reduced the number of apoptotic cells as shown by the TUNEL assay. Levels of TRAF6, NF-κB p65, ASC, NLRP3, caspase-1, and pro-caspase-1 proteins were increased after ischemia-reperfusion (I/R) but were reversed by DADLE. Immunofluorescence staining results for NF-κB and NLRP3 demonstrated similar changes. ELISA assays showed that TNF-α and IL-1β concentrations were increased in the model and reversed by DADLE. Conclusion: DADLE can significantly ameliorate myocardia ischemia-reperfusion injury (MIRI), with the dosage of 0.5 mg/kg presenting the greatest benefit. DADLE may exert its protective effects by activating the TRAF6/NF-κB/NLRP3 signaling pathway.